WIXLIB

Figure 5. The standard curve for the amplification of the c-myc target detected using aFAM-labeled probe.Calculating the Input AmountPerform the following steps to calculate the input amount for unknown samples.Step1ActionFor the line shown in Figure 5, calculate the log input amount by entering thefollowing formula in one cell of the work sheet: ([cell containing CT value] –b)/mb y-intercept of standard curve linem slope of standard curve lineNote2In Figure 5, b 25.712 and m -3.385 for the equation y mx b.Calculate the input amount by entering the following formula in an adjacent cell: 10 [cell containing log input amount]NoteThe units of the calculated amount are the same as the units used toconstruct the standard curve, which are nanograms of Total Raji RNA. If it iscalculated that an unknown has 0.23 ng of Total Raji RNA, then the samplecontains the same amount of c-myc mRNA found in 0.23 ng of the Raji ControlRNA.Page 8 of 363Repeat the steps to construct a standard curve for the endogenous reference usingthe CT values determined with the JOE-labeled GAPDH probe. Refer to Table 1 onpage 10.4Because c-myc and GAPDH are amplified in separate tubes, average the c-mycand GAPDH values separately.5Divide the amount of c-myc by the amount of GAPDH to determine the normalizedamount of c-myc (c-mycN).User Bulletin #2: ABI PRISM 7700 Sequence Detection System

Comparing Samples with a CalibratorThe normalized amount of target (c-mycN) is a unitless number that can be used tocompare the relative amount of target in different samples. One way to make thiscomparison is to designate one of the samples as a calibrator. In Table 1 on page 10,brain is designated as the calibrator; brain is arbitrarily chosen because it has thelowest expression level of the target.Relative Standard Curve ResultsEach c-mycN value in Table 1 is divided by the brain c-mycN value to give the values inthe final column. These results indicate the kidney sample contains 5.5 as muchc-myc mRNA as the brain sample, liver 34.2 as much, and lung 15.7 as much.Perform the following steps to determine relative values.StepAction1Average the c-myc and GAPDH values from Table 1.2Divide the c-myc average by the GAPDH average.3Designate the calibrator.4Divide the averaged sample value by the averaged calibrator value.User Bulletin #2: ABI PRISM 7700 Sequence Detection SystemPage 9 of 36

Table 1. Amounts of c-myc and GAPDH in Human Brain, Kidney, Liver, and gAveragec-mycng Total Raji RNAGAPDHng Total Raji 0400.520.039 0.0040.54 .960.41 0.0161.02 .270.70 0.0360.28 .800.93 0.0440.81 0.041c-mycNNorm. to GAPDHac-mycNRel. to Brainb0.07 0.0081.0 0.120.40 0.0255.5 0.352.49 0.17334.2 2.371.15 0.07915.7 1.09a. The c-mycN value is determined by dividing the average c-myc value by the average GAPDH value. Thestandard deviation of the quotient is calculated from the standard deviations of the c-myc and GAPDH values.See “Standard Deviation Calculation Using the Standard Curve Method” on page 34.b. The calculation of c-mycN relative to brain involves division by the calibrator value. This is division by anarbitrary constant, so the cv of this result is the same as the cv for c-mycN.Page 10 of 36User Bulletin #2: ABI PRISM 7700 Sequence Detection System

Comparative CT Method (Separate Tubes)Similar to Standard The comparative CT method is similar to the standard curve method, except it usesCurve Method arithmetic formulas to achieve the same result for relative quantitation.NoteIt is possible to eliminate the use of standard curves for relative quantitation as long asa validation experiment is performed. See “Validation Experiment” on page 14.Arithmetic Formulas The amount of target, normalized to an endogenous reference and relative to acalibrator, is given by:2 – CTDerivation of the FormulaThe equation that describes the exponential amplification of PCR is:Xn Xo ( 1 EX ) nwhere:Xn number of target molecules at cycle nXo initial number of target moleculesEX efficiency of target amplificationn number of cyclesThe threshold cycle (CT) indicates the fractional cycle number at which the amount ofamplified target reaches a fixed threshold. Thus,X T X o ( 1 E X ) CT, X K Xwhere:XT threshold number of target moleculesCT,X threshold cycle for target amplificationKX constantUser Bulletin #2: ABI PRISM 7700 Sequence Detection SystemPage 11 of 36

A similar equation for the endogenous reference reaction is:R T R o ( 1 E R ) CT, R K Rwhere:RT threshold number of reference moleculesRo initial number of reference moleculesER efficiency of reference amplificationCT, R threshold cycle for reference amplificationKR constantDividing XT by RT gives the following expression:CX o ( 1 E X ) T, X K XXT- ------- K------- ------------------------------------------CRTR o ( 1 E R ) T, R K RThe exact values of XT and RT depend on a number of factors, including: Reporter dye used in the probe Sequence context effects on the fluorescence properties of the probe Efficiency of probe cleavage Purity of the probe Setting of the fluorescence threshold.Therefore, the constant K does not have to be equal to one.Assuming efficiencies of the target and the reference are the same:EX ER E,XoC T, X – C T, R------ ( 1 E ) KRoorXN ( 1 E ) C T Kwhere:Page 12 of 36XN Xo/Ro, the normalized amount of target CT CT,X - CT,R, the difference in threshold cycles for target and referenceUser Bulletin #2: ABI PRISM 7700 Sequence Detection System

Rearranging gives the following expression:XN K ( 1 E )– C TThe final step is to divide the XN for any sample q by the XN for the calibrator (cb):– CX– C TK ( 1 E ) T, qN, q- ( 1 E )-------------- ------------------------------------------– CX N, cbK ( 1 E ) T, cbwhere: CT CT,q – CT,cbFor amplicons designed and optimized according to Applied Biosystems guidelines(amplicon size 150 bp), the efficiency is close to one. Therefore, the amount oftarget, normalized to an endogenous reference and relative to a calibrator, is given by:2 – CTRelative Efficiency of For the CT calculation to be valid, the efficiency of the target amplification and theTarget and efficiency of the reference amplification must be approximately equal. A sensitiveReference method for assessing if two amplicons have the same efficiency is to look at how CTvaries with template dilution. The standard curves for c-myc and GAPDH used in theprevious section provide the necessary data. Table 2 shows the average CT value forc-myc and GAPDH at different input amounts.Table 2. Average CT Value for c-myc and GAPDH at Different Input AmountsInput Amountng Total RNAc-mycAverage CTGAPDHAverage CT CTc-myc – GAPDH1.025.59 0.0422.64 0.032.95 0.050.526.77 0.0923.73 0.053.04 0.100.228.14 0.0525.12 0.103.02 0.110.129.18 0.1326.16 0.023.01 0.130.0530.14 0.0327.17 0.062.97 0.070.0231.44 0.1628.62 0.102.82 0.190.0132.42 0.1229.45 0.082.97 0.14Figure 6 on page 14 shows a plot of log input amount versus CT. If the efficiencies ofthe two amplicons are approximately equal, the plot of log input amount versus CThas a slope of approximately zero.User Bulletin #2: ABI PRISM 7700 Sequence Detection SystemPage 13 of 36

Validation Before using the CT method for quantitation, perform a validation experiment likeExperiment that in Figure 6 to demonstrate that efficiencies of target and reference areapproximately equal. The absolute value of the slope of log input amount vs. CTshould be 0.1. The slope in Figure 6 is 0.0492, which passes this test. Once this isproven, you can use the CT calculation for the relative quantitation of target withoutrunning standard curves on the same plate.If the efficiencies of the two systems are not equal, perform quantitation using thestandard curve method. Alternatively, new primers can be designed and synthesizedfor the less efficient system to try to boost efficiency.Figure 6. Plot of log input amount versus CTPage 14 of 36User Bulletin #2: ABI PRISM 7700 Sequence Detection System

Comparative CT The CT data used to determine the amounts of c-myc and GAPDH mRNA shown inResults Table 1 on page 10 are used to illustrate the CT calculation. Table 3 shows theaverage CT results for the human brain, kidney, liver, and lung samples and how theseCTs are manipulated to determine CT, CT, and the relative amount of c-mycmRNA. The results are comparable to the relative c-myc levels determined using thestandard curve method.Table 3.Relative Quantitation Using the Comparative CT MethodTissuec-mycAverage CTGAPDHAverage CTBrain30.49 0.1523.63 0.096.86 0.170.00 0.171.0(0.9–1.1)Kidney27.03 0.0622.66 0.084.37 0.10–2.50 0.105.6(5.3–6.0)Liver26.25 0.0724.60 0.071.65 0.10–5.21 0.1037.0(34.5–39.7)Lung25.83 0.0723.01 0.072.81 0.10–4.05 0.1016.5(15.4–17.7) CT CTc-mycN CT, Brainb Rel. to Braincc-myc–GAPDHa CT– a. The CT value is determined by subtracting the average GAPDH CT value from the average c-myc CTvalue. The standard deviation of the difference is calculated from the standard deviations of the c-myc andGAPDH values. See “Standard Deviation Calculation Using the Comparative Method” on page 35.b. The calculation of CT involves subtraction by the CT calibrator value. This is subtraction of an arbitraryconstant, so the standard deviation of CT is the same as the standard deviation of the CT value.c. The range given for c-mycN relative to brain is determined by evaluating the expression: 2 – CTwith CT s and CT – s, where s the standard deviation of the CT value.User Bulletin #2: ABI PRISM 7700 Sequence Detection SystemPage 15 of 36

Multiplex PCR (Same Tube)Overview Multiplex PCR is the use of more than one primer pair in the same tube. You can usethis method in relative quantitation where one primer pair amplifies the target andanother primer pair amplifies the endogenous reference in the same tube. You canperform a multiplex reaction for both the standard curve method and the comparativemethod.Dyes Available for The availability of multiple reporter dyes for TaqMan probes makes it possible toTaqMan Probes detect amplification of more than one target in the same tube. The reporter dyescurrently recommended for probes are 6-FAM, TET, and JOE. These dyes aredistinguishable from one another because they have different emission wavelengthmaxima: 6-FAM, λmax 518 nm TET, λmax 538 nm JOE, λmax 554 nmMulticomponenting The ABI PRISM 7700 Sequence Detection System software uses a process calledmulticomponenting to distinguish reporter dyes, the quencher dye TAMRA (λmax 582nm), and the passive reference dye ROX (λmax 610 nm). Multicomponenting is amathematical algorithm that uses pure dye reference spectra to calculate thecontribution of each dye to a complex experimental spectrum.Accurate Because of experimental variation in measuring both the reference spectra and theQuantitation sample spectra, multicomponenting introduces some error into the determination ofeach dye’s contribution. The degree of error depends on how well the various dyes arespectrally resolved. The greater the spectral overlap between two dyes, the greaterthe error. Thus, for the most accurate quantitation using two probes in one tube, usethe reporter dyes that have the largest difference in emission maximum: 6-FAM andJOE.The TaqMan GAPDH Control Reagents (P/N 402869) provide a JOE-labeled probe forhuman GAPDH mRNA. Therefore, when using GAPDH as an endogenous reference,label the probe for the target mRNA with 6-FAM.Avoiding Reactions to amplify two different segments in the same tube share commonCompetition in reagents. If the two segments have different initial copy numbers, it is possible for theReactions more abundant species to use up these common reagents, impairing amplification ofthe rarer species. For accurate quantitation, it is important that the two reactions donot compete. Competition can be avoided by limiting the concentration of primersused in the amplification reactions.Page 16 of 36User Bulletin #2: ABI PRISM 7700 Sequence Detection System

Limiting Primer Figure 7 shows PCR amplifications with decreasing concentrations of primers. AtConcept 120 and 80 nM, the amplification plots are similar, indicating that the reactions are notlimited by the amount of primers. The remaining plots show that the more dilute theprimers, the lower the plateau fluorescence level at the end of the reaction. Thisdemonstrates that a lower primer concentration limits the reaction, forcing it to plateauat a lower level of product.Figure 7. PCR amplifications with decreasing primer concentrationsIn terms of kinetic analysis, however, all the reactions except 4 nM have the same CTvalue. The strategy for performing two independent reactions in the same tube is toadjust the primer concentrations such that accurate CTs are obtained, but soon afterthat, the exhaustion of primers defines the end of the reaction. In this way,amplification of the majority species is stopped before it can limit the commonreactants available for amplification of the minority species.Considering Relative Abundance of the Target and ReferenceIn applying the limiting primer concept to target and endogenous referenceamplification, the relative abundance of the two species must be considered. Forquantitation of gene expression, it is possible to use rRNA as an endogenousreference. The concentration of rRNA in total RNA is always greater than theconcentration of any target mRNA. Therefore, in multiplex reactions amplifying bothtarget and rRNA, only the concentrations of the rRNA primers need to be limited. Forc-myc and GAPDH, it is not known if the abundance of one RNA is always greaterthan the other in the tissues and cell lines that might be examined. For amplifyingc-myc and GAPDH in the same tube, limiting primer concentrations need to bedefined for both amplicons.Defining Limiting Primer ConcentrationsDefine limiting primer concentrations by running a matrix of forward and reverseprimer concentrations. The desired concentrations are those that show a reduction in Rn but little effect on CT. Figure 8 on page 18 and Figure 9 on page 19 show theresults when GAPDH is amplified using all combinations of forward and reverseprimers at 80, 40, 30, and 20 nM.User Bulletin #2: ABI PRISM 7700 Sequence Detection SystemPage 17 of 36

Figure 8. GAPDH amplified using all combinations of forward and reverse primersThe CT results in Figure 8 show that the CT value using 30 nM each primer is thesame as 80 nM each primer. Figure 9 shows that the Rn at 30 nM each primer isreduced relative to more concentrated primers. Thus, by amplifying GAPDH with30 nM each primer, accurate CTs are obtained, but the GAPDH reaction is shut downbefore it affects amplification of a less abundant species. In order to provide a marginfor error, a concentration of 40 nM each GAPDH primer is used in the “Multiplex PCRExample” on page 19.Page 18 of 36User Bulletin #2: ABI PRISM 7700 Sequence Detection System

Figure 9. GAPDH uplifted using all combinations of forward and reverse primersA similar experiment defines 50 nM each primer as limiting primer conditions foramplification of c-myc. In these primer limitation studies, the buffer and thermalcycling conditions are the same for the two systems run in the same tube. Thisprocess is simplified by using a two-step RT-PCR protocol, because the PCR can beoptimized separately from the reverse transcriptase reaction. This allows you to useour Assay Design Guidelines for DNA amplification when they become available(currently in progress). These generate primers and probes that work well using ageneric set of buffer and thermal cycling conditions.NoteIf limiting primer concentrations cannot be found, quantitation can still be obtained byrunning the reactions in separate tubes. Alternatively, the primers can be redesigned andretested to find limiting concentrations. The primers generally need to be altered by increasingtheir length one or two nucleotides in order to increase their TMs.Multiplex PCR The experiment quantitating the target c-myc normalized to the endogenousExample reference GAPDH is repeated running both amplifications in the same tube. SeeFigure 1 on page 4 for the setup on the FAM layer and see Figure 10 on page 20 forthe setup on the JOE layer. Figure 10 is similar to the setup in Figure 2 on page 5,except GAPDH is being amplified in rows A–D (the same tubes where amplification ofc-myc is being performed). This illustrates one advantage of performing target andreference reaction in the same tube—higher throughput.Higher throughput is most evident if you are interested in analyzing a single targetbecause the number of sample tubes is reduced by a factor of two. As the number oftargets analyzed on the same plate increases, the advantage of same tube overseparate tube decreases, because a single set of reference reactions can be used tonormalize all of the different target reactions.User Bulletin #2: ABI PRISM 7700 Sequence Detection SystemPage 19 of 36

Figure 10. Plate setup for relative quantitation of the c-myc mRNA on JOE layerSpectral Compensation FeatureWhen analyzing data that have two reporters in the same tube, use the specialsoftware feature called Spectral Compensation. This is an enhancement of themulticomponenting algorithm because it provides improved well-to-well spectralresolution for multi-reporter applications. However, it can also be a liability because itincreases noise of the fluorescence measurements.With multiple reporter dyes in the same tube, Spectral Compensation should beturned on because accurate separation of dye signals is more important thanincreased precision. When one reporter dye is used in a tube, Spectral Compensationshould be left off in order to benefit from the improved precision.Page 20 of 36User Bulletin #2: ABI PRISM 7700 Sequence Detection System

Perform the following steps to access Spectral Compensation.StepAction1Under Diagnostics in the Instruments menu, select the Advanced Options dialogbox.2To analyze more than one reporter dye in the same tube, check the box markedUse Spectral Compensation for Real Time.3Click OK.NoteIgnore the warning message if the only change made is to turn SpectralCompensation on or off.Data HandlingAfter the analysis is performed, setting the baselines and thresholds, exporting thedata to Excel, and drawing standard curves in Excel are exactly the same as in theseparate tube example. For both the standard curve and CT method, the onlydifference between same-tube and separate-tube analysis is how replicates areaveraged.Multiplex PCR Results (Standard Curve Method)Table 4 on page 22 shows the results of the same-tube experiment using the standardcurve method. Both the c-myc and GAPDH amounts are determined from a sin

User Bulletin #2: ABI P RISM 7700 Sequence Detection System Page 7 of 36 Constructing a Relative Standard Curve The ABI P RISM 7700 Sequence Detection System (version 1.6 software) is not designed to construct two standard curves on the same plate. To analyze this experiment, Results are exported to an Excel spreadsheet by choosing the Export